甲床细胞体外培养的实验研究

Experimental Study on Cultivation of Cells of Nail Beds

目的探讨甲床上皮细胞及成纤维细胞体外培养的可行性。方法取20周以上引产胎儿的甲床,采用组织块培养法进行原代培养获得甲床上皮细胞和成纤维细胞,观察细胞形态,进行HE染色,免疫组织化学染色对细胞进行鉴定,甲床上皮细胞检测皮肤型角蛋白17,成纤维细胞检测波形蛋白。结果甲床上皮细胞培养2～3 d从组织块中游出,并在组织块周围形成生长晕,13 d左右细胞生长密度可达瓶底的70%～80%,扁圆形细胞,呈铺路石样排列。70%以上特异性角蛋白17抗体染色阳性,证明培养的细胞为甲床上皮细胞;甲床成纤维细胞培养3 d观察可见组织块周围有许多生长的细胞,细胞呈现圆或长梭形、等成纤维细胞的特征,胞体丰富,胞质均匀,细胞核清晰可见,11 d左右细胞生长密度可达瓶底的70%～80%。培养甲床成纤维细胞抗波形蛋白抗体染色阳性,说明培养的细胞为成纤维细胞。结论通过组织块法成功培养出甲床上皮细胞及成纤维细胞,为组织工程甲床的深入研究奠定了基础。

Objective To discuss the practicability of in vitro culture of epithelias and desmocytes of nail beds.Methods Take nail beds of fetus over 20 weeks and get epithelia and desmocytes of nail beds by tissue mass culture method through primary culture.View the cell shape,HE dye,and identity the cells through immunohistochemical staining（skin type keratin-17 for epithelia of nail beds and waveform protein for desmocytes）.Results Epithelias of nail beds swam out from the tissue block after 2 to 3 days in culture and shape outgrowth around the tissue block.Growth density of the cells can reach 70% to 80% of the bottle in about 13 days and oblate cells forming like stepping stones.More than 70% specific keratin-17 staining positive proving the cells in culture are epithelia of nail beds.Many growing cell can be observed around the tissue block after 3 days of desmocytes of nail beds in culture.The cells show characteristics of round or long spindle,plentiful,cytoplasm well distributed and the karyon can be seen clearly.Growth density of the cells can reach 70% to 80% of the bottle in about 11 days and the waveform protein antibody staining positive proves the cells in culture are desmocytes of nail beds.Conclusion Epithelia and desmocytes of nail beds are successfully obtained through tissue mass culture method,which lays the basis of deeper research on tissue engineering nail beds.